Faculty Opinions recommendation of Measurement of membrane binding between recoverin, a calcium-myristoyl switch protein, and lipid bilayers by AFM-based force spectroscopy.

Perforin-like proteins (PLPs) play key roles in the mechanisms associated with parasitic disease caused by apicomplexans such as Plasmodium (malaria) and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1; PvPLP1 is important for invasion of mammalian hosts by the parasite and establishment of a chronic infection, PvPLP2 is important during the symptomatic blood stage of the parasite life cycle. Determination of the crystal structure of the membrane-binding APCβ domain of PvPLP1 reveals notable differences with that of TgPLP1, which are reflected in its inability to bind lipid bilayers in the way that TgPLP1 and PvPLP2 can be shown to. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow a dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal a similar tropism for lipids found enriched in the inner leaflet of the mammalian plasma membrane. In addition to this shared mode of membrane binding PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APCβ domains of Apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins, in their different developmental roles within parasite life cycles.

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Influence of Cholesterol on the Phase Transition of Lipid Bilayers: A Temperature-Controlled Force Spectroscopy Study

Langmuir ◽

10.1021/la302620t ◽

2012 ◽

Vol 28 (35) ◽

pp. 12851-12860 ◽

Cited By ~ 107

Author(s):

Lorena Redondo-Morata ◽

Marina I. Giannotti ◽

Fausto Sanz

Keyword(s):

Phase Transition ◽

Lipid Bilayers ◽

Force Spectroscopy ◽

Spectroscopy Study ◽

Temperature Controlled

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Bax-type Apoptotic Proteins Porate Pure Lipid Bilayers through a Mechanism Sensitive to Intrinsic Monolayer Curvature

Journal of Biological Chemistry ◽

10.1074/jbc.m206069200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49360-49365 ◽

Cited By ~ 171

Author(s):

Gorka Basañez ◽

Juanita C. Sharpe ◽

Jennifer Galanis ◽

Teresa B. Brandt ◽

J. Marie Hardwick ◽

...

Keyword(s):

Lipid Bilayers ◽

Mitochondrial Membrane ◽

Pore Formation ◽

Membrane Binding ◽

Membrane Permeabilization ◽

Outer Mitochondrial Membrane ◽

Intrinsic Curvature ◽

Differential Effects ◽

Cleavage Fragment ◽

Induced Changes

During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and ΔN76Bcl-xL(the Bax-like cleavage fragment of Bcl-xL) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or ΔN76Bcl-xL. Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.

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Single-molecule force spectroscopy of protein-membrane interactions

10.1101/170290 ◽

2017 ◽

Author(s):

Lu Ma ◽

Yiying Cai ◽

Yanghui Li ◽

Junyi Jiao ◽

Zhenyong Wu ◽

...

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Membrane Binding ◽

Force Spectroscopy ◽

Binding Energies ◽

Membrane Interactions ◽

Spatiotemporal Resolution ◽

Single Molecule Force Spectroscopy ◽

C2 Domains ◽

Protein Membrane

AbstractMany biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

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Discovery of novel membrane binding structures and functions

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0074 ◽

2014 ◽

Vol 92 (6) ◽

pp. 555-563 ◽

Cited By ~ 17

Author(s):

Irina Kufareva ◽

Marc Lenoir ◽

Felician Dancea ◽

Pooja Sridhar ◽

Eugene Raush ◽

...

Keyword(s):

Membrane Protein ◽

Lipid Bilayers ◽

Protein Interactions ◽

Membrane Binding ◽

Three Dimensional ◽

Intrinsic Activity ◽

Resonance Spectroscopy ◽

Pleckstrin Homology ◽

Lysobisphosphatidic Acid ◽

Membrane Interactive

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome.

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A myristoyl switch regulates membrane binding of HIV-1 Gag

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0308043101 ◽

2004 ◽

Vol 101 (2) ◽

pp. 417-418 ◽

Cited By ~ 62

Author(s):

M. D. Resh

Keyword(s):

Membrane Binding ◽

Myristoyl Switch ◽

Hiv 1

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Single-molecule force spectroscopy of protein-membrane interactions

eLife ◽

10.7554/elife.30493 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 22

Author(s):

Lu Ma ◽

Yiying Cai ◽

Yanghui Li ◽

Junyi Jiao ◽

Zhenyong Wu ◽

...

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Membrane Binding ◽

Force Spectroscopy ◽

Binding Energies ◽

Membrane Interactions ◽

Spatiotemporal Resolution ◽

Single Molecule Force Spectroscopy ◽

C2 Domains ◽

Protein Membrane

Many biological processes rely on protein–membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2–7 pN and had binding energies of 4–14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein–membrane interactions.

Download Full-text